Cell removal method, cell removal system, and white blood cell removal method

ABSTRACT

In the removal of white blood cells from blood, the efficiency of white blood cell removal by a filter is improved. Centrifugal force is applied to blood placed in a blood bag by centrifugal separation to form a plurality of separation layers having a concentration gradient of white blood cells. The separation layers are passed through a filter in ascending order of white blood cell concentration to remove the white blood cells from the separation layers. The separation layers from which the white blood cells have been removed are received in a first blood component bag.

BACKGROUND

The present invention relates to a cell removal method, a cell removalsystem, and a white blood cell removal method.

In order to perform blood component transfusion, for example, it isnecessary to separate blood components such as packed red blood cellcomponents or plasma components from blood to produce a blood componentproduct. A blood component product is produced using, for example, asystem that can be mounted on a centrifugal separator. Such a systemincludes, for example, a blood collection bag that receives bloodcollected from the human body, a blood bag, a blood component bag thatreceives separated blood components, a tube that connects them, and thelike (see Patent Literature 1).

In the production of a blood component product, for example, blood inthe blood collection bag is passed through a filter using the differencein gravity to remove white blood cells that are a pathogenic substance,and then received in the blood bag. Subsequently, the blood componentproduction system is mounted on a centrifugal separator. Using thecentrifugal separator, blood in the blood bag is separated bycentrifugation into a plasma component layer and a red blood cellcomponent layer, for example, according to the difference in cellconcentration. Subsequently, for example, the system is mounted on anapheresis system, and the plasma component layer in the blood bag ispressed out and received in the blood component bag to produce a plasmaproduct.

In addition, the red blood cell component layer remaining in the bloodbag is pressed out by the apheresis system and received in another bloodcomponent bag to produce a red blood cell product.

Patent Literature 1: Patent Publication JP-A-2009-90136

Patent Literature 2: Patent Publication JP-A-2002-320669

Incidentally, for example, in the production of a blood componentproduct mentioned above, in order to more reliably prevent side effectsof blood component transfusion caused by white blood cells, etc., it isnecessary to further increase the efficiency of white blood cell removalby a filter. In addition, by increasing the efficiency of white bloodcell removal by a filter, the filter can be reduced in size. By reducingthe filter size, the amount of useful components caught by the filterand wasted, such as red blood cells, can be reduced.

SUMMARY

The present invention has been accomplished against the abovebackground. An object of the present invention is to increase theefficiency of cell removal by a filter in the removal of predeterminedcells such as white blood cells from a body fluid such as blood.

The present invention for achieving the above object is a cell removalmethod for removing predetermined cells from a body fluid. The methodincludes: applying centrifugal force to a body fluid placed in a firstbag by centrifugal separation to form separation layers having aconcentration gradient of the predetermined cells; passing theseparation layers through a filter in ascending order of theconcentration of the predetermined cells according to the concentrationgradient of the predetermined cells to remove the predetermined cellsfrom the separation layers; and receiving the separation layers fromwhich the predetermined cells have been removed in a second bag.Incidentally, the concentration of predetermined cells herein is massconcentration.

According to the present invention, by centrifugal force, separationlayers having a concentration gradient of predetermined cells areformed, and the separation layers having a cell concentration gradientare passed through a filter in ascending order of the concentration ofthe predetermined cells. Thus, the separation layer having the highestconcentration of the predetermined cells can be the last to pass throughthe filter. Accordingly, filtration can be continued while suppressing adecrease in the predetermined cell removal performance caused by atemporary decrease in the number of adsorption sites accompanying theprogress of filtration, which is seen in the case where a body fluidcomponent having a uniform cell concentration is passed through afilter. As a result, as compared with such a case, the efficiency ofpredetermined cell removal by a filter can be increased.

It is also possible that the separation layers are passed through thefilter using the centrifugal force by the centrifugal separation. Insuch a case, the separation layers can be passed through the filterwhile reliably maintaining the separated state of the separation layers.In addition, the separation layers can be passed through the filterduring or immediately after the centrifugal separation that separatesthe body fluid into the separation layers, whereby the time of the cellremoval process can be shortened. In addition, the need for a specialdevice for passing a body fluid component through a filter iseliminated, whereby the cost of the cell removal process can be reduced.

It is also possible that when the separation layers are passed throughthe filter, a passing rate may be changed according to the concentrationof the predetermined cells. In such a case, according to the cellconcentration of each separation layer, the separation layers can bepassed at an optimal rate, whereby the performance of the filter iseffectively exhibited, and the efficiency of predetermined cell removalby the filter can be improved.

It is also possible that a body fluid component produced by removing thepredetermined cells from the separation layers is received in the secondbag, and that a preservation liquid for preserving the body fluidcomponent may be added to the separation layers before being passedthrough the filter. In such a case, the preservation liquid can be addedin the process of removing the predetermined cells. Therefore, there isno need for the process of separately adding a preservation liquid. Inaddition, the separation layers are diluted with the preservation liquidand thus more easily pass through the filter. Therefore, the burden onthe filter can be reduced, prolonging the life of the filter.

It is also possible that an additional separation layer other than theseparation layers that is separated by the centrifugal force describedabove is received in a third bag separately from the separation layersdescribed above.

The predetermined cells may be pathogens. In addition, it is alsopossible that the body fluid is blood, the predetermined cells are whiteblood cells, and the predetermined cells are removed from the separationlayers to produce a blood component containing red blood cells as a maincomponent.

The present invention from another point of view is a cell removalsystem that is mounted on a centrifugal separator and removespredetermined cells from a body fluid. The cell removal system includes:a body fluid bag that receives a body fluid; a first body fluidcomponent bag that receives a first body fluid component having arelatively high cell concentration in the body fluid; a second bodyfluid component bag that receives a second body fluid component having arelatively low cell concentration in the body fluid; a first channelthat connects the first body fluid component bag and the body fluid bag;a second channel that connects the second body fluid component bag andthe body fluid bag; a filter that is provided in the first channel andremoves predetermined cells from a body fluid component passing throughthe channel; a first on-off valve that opens and closes the firstchannel; and a second on-off valve that opens and closes the secondchannel. The first channel and the second channel are connected to anend portion of the body fluid bag, the end portion being, when mountedon the centrifugal separator, on the side of a direction of centrifugalforce by the centrifugal separator.

According to the present invention, the first channel and the secondchannel are connected to an end portion of the body fluid bag on theside of the direction of centrifugal force. Therefore, the separationlayers having a concentration gradient of predetermined cells separatedalong the direction of centrifugal force by the centrifugal separatorcan be passed through the filter in order of closeness to the firstchannel. Accordingly, the separation layers can be passed through thefilter in ascending order of the concentration of the predeterminedcells. As a result, as compared with the case where a body fluidcomponent having a uniform concentration of cells to be removed ispassed through a filter, the efficiency of cell removal by a filter canbe increased. In addition, a separation layer containing the second bodyfluid component other than the separation layers containing the firstbody fluid component can also be received in the second body fluidcomponent bag using the second channel. In addition, because the firstchannel and the second channel are connected to an end portion of thebody fluid bag on the side of the direction of centrifugal force, usingthe centrifugal force of the centrifugal separator, it is possible notonly to separate the body fluid in the body fluid bag, but also to senda fluid to the body fluid component bag using the first channel or thesecond channel. Accordingly, the processing time from the body fluidseparation to the body fluid component production can be shortened.

The present invention from still another point of view is a cell removalsystem that is mounted on a centrifugal separator and removespredetermined cells from a body fluid. The cell removal system includes:a body fluid bag that receives a body fluid; a first body fluidcomponent bag that receives a first body fluid component having arelatively high cell concentration in the body fluid; a second bodyfluid component bag that receives a second body fluid component having arelatively low cell concentration in the body fluid; a first channelthat connects the first body fluid component bag and the body fluid bag;a second channel that connects the second body fluid component bag andthe body fluid bag; a filter that is provided in the first channel andremoves predetermined cells from a body fluid component passing throughthe channel; a first on-off valve that opens and closes the firstchannel; and a second on-off valve that opens and closes the secondchannel. The first channel is connected to an end portion of the bodyfluid bag, the end portion being, when mounted on the centrifugalseparator, on the side of a direction of centrifugal force by thecentrifugal separator. The second channel is connected to an end portionof the body fluid bag, the end portion being, when mounted on thecentrifugal separator, on the opposite side from the direction ofcentrifugal force by the centrifugal separator.

According to the present invention, the first channel is connected to anend portion of the body fluid bag on the side of the direction ofcentrifugal force. Therefore, the separation layers having aconcentration gradient of predetermined cells separated along thedirection of centrifugal force by the centrifugal separator can bepassed through the filter in order of closeness to the first channel.Accordingly, the separation layers can be passed through the filter inascending order of the concentration of the predetermined cells. As aresult, as compared with the case where a body fluid component having auniform concentration of cells to be removed is passed through a filter,the efficiency of cell removal by a filter can be increased. Inaddition, a separation layer containing the second body fluid componentother than the separation layers containing the first body fluidcomponent can also be received in the second body fluid component bagusing the second channel. In addition, because the first channel isconnected to an end portion of the body fluid bag on the side of thedirection of centrifugal force, using the centrifugal force of thecentrifugal separator, it is possible not only to separate the bodyfluid in the body fluid bag, but also to send the separation layers tothe first body fluid component bag using the first channel. Accordingly,the processing time from the body fluid separation to the body fluidcomponent production can be shortened.

It is also possible that an additional filter may be provided in thesecond channel to remove predetermined cells from a body fluid componentpassing through the channel. In such a case, predetermined cells can beremoved also from a body fluid component having a low cellconcentration.

It is also possible that the cell removal system further includes apreservation liquid supply circuit that is provided in the first channelin a position closer to the body fluid bag than the filter and suppliesa preservation liquid for preserving the first body fluid component. Insuch a case, the preservation liquid can be supplied together with theremoval of predetermined cells. In addition, a body fluid componentpassing through the first channel is diluted with the preservationliquid and thus more easily passes through the filter. Therefore, theburden on the filter can be reduced, prolonging the life of the filter.

The predetermined cells may be a disease-causing substance. In addition,it is also possible that the body fluid is blood, the first body fluidcomponent is a blood component containing red blood cells as a maincomponent, the second body fluid component is a blood componentcontaining plasma as a main component, and the predetermined cells maybe white blood cells.

The present invention from still another point of view is a method forremoving white blood cells from a body fluid containing red blood cellsand white blood cells. The method includes: applying centrifugal forceto a body fluid placed in a first bag by centrifugal separation to formseparation layers containing at least the red blood cells and the whiteblood cells and having a concentration gradient of the white bloodcells; passing the separation layers through a filter in ascending orderof the concentration of the white blood cells according to theconcentration gradient of the white blood cells to remove the whiteblood cells from the separation layers; and receiving the separationlayers from which the white blood cells have been removed in a secondbag.

In the white blood cell removal method, when the separation layers arepassed through the filter, a passing rate may be changed according tothe concentration of the white blood cells.

According to the present invention, in the removal of predeterminedcells from a body fluid, the efficiency of predetermined cell removal bya filter can be improved. In addition, the improvement of the efficiencyof predetermined cell removal allows for the reduction of filter size.As a result, the amount of useful components caught by the filter andwasted can be reduced, and the recovery of useful body fluid componentscan be increased.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanatory drawing schematically showing the configurationof a cell removal system.

FIG. 2 is a longitudinal sectional view schematically showing theconfiguration of a centrifugal separator.

FIG. 3 is a plan view schematically showing the configuration of acentrifugal separator.

FIG. 4 is an explanatory drawing showing the configuration of a pressingmechanism.

FIG. 5 is an explanatory drawing of a cell removal system in the casewhere a first channel and a second channel are connected to a first endportion of a blood bag.

FIG. 6 is an explanatory drawing of a cell removal system in the casewhere a filter is provided in a second channel.

FIG. 7 is an explanatory drawing of a cell removal system in the casewhere a preservation liquid supply circuit is connected to a firstchannel.

FIG. 8 is an explanatory drawing showing the case where a blood bag isinstalled in a centrifugal separator in a different direction.

FIG. 9 is an explanatory drawing of a cell removal system in the case ofthe production of a whole blood product.

DETAILED DESCRIPTION

Hereinafter, a preferred embodiment of the present invention will bedescribed with reference to the drawings. FIG. 1 is an explanatorydrawing schematically showing the configuration of a cell removal system1 according to this embodiment.

The cell removal system 1 can be mounted on a centrifugal separator 2described below and includes, for example, a blood bag 10 as a firstbag, a first blood component bag 11 as a second bag, a second bloodcomponent bag 12 as a third bag, a first channel 13, a second channel14, a filter 15, etc.

The blood bag 10 is made of soft resin, etc., for example, and isdeformable. The blood bag 10 has connected thereto a channel 20 forblood collection, and can receive blood collected from the human body.The channel 20 for blood collection has provided therein an on-off valve21 that opens and closes the channel 20.

The first blood component bag 11 and the second blood component bag 12are made of soft resin, etc., for example, and are deformable.

As the first channel 13 and the second channel 14, soft tubes are used,for example. The first channel 13 connects the blood bag 10 and thefirst blood component bag 11. The first channel 13 is connected to, forexample, a first end portion 10 a of the blood bag 10. The end portion10 a is on the side of the direction of centrifugal force F by thecentrifugal separator 2 when the blood bag 10 is mounted on thecentrifugal separator 2. The first channel 13 has provided therein afirst on-off valve 30 that opens and closes the channel 13.

The second channel 14 connects the blood bag 10 and the second bloodcomponent bag 12. The second channel 14 is connected to, for example, asecond end portion 10 b of the blood bag 10. The end portion 10 b is onthe opposite side from the direction of centrifugal force F by thecentrifugal separator 2 when the blood bag 10 is mounted on thecentrifugal separator 2. The second channel 14 has provided in a secondon-off valve 31 that opens and closes the channel 14. Incidentally, thechannel 20 for blood collection mentioned above is also connected to thesecond end portion 10 b of the blood bag 10.

The filter 15 is provided in the first channel 13. The filter 15 isformed of a porous body, for example, which can filter out white bloodcells as predetermined cells, which are pathogens. The filter 15 isprovided between the first on-off valve 30 and the first blood componentbag 11.

The centrifugal separator 2 includes, for example, as shown in FIG. 2and FIG. 3, in a housing 50, a rotary actuators 51 such as a motor, arotary container 52 which is rotated by the rotary actuator 51 and onwhich a plurality of cell removal systems 1 can be mounted, etc. In therotary container 52, for example, the blood bag 10 of the cell removalsystem 1 can be installed in such a manner that the first end portion 10a, to which the first channel 13 is connected, faces the outside (theside of the direction of centrifugal force F), while the second endportion 10 b, to which the second channel 14 is connected, faces thecenter (the opposite side from the direction of centrifugal force F).Incidentally, in this embodiment, the blood bag 10 is installed in sucha manner that the length direction of the bag (horizontal direction inFIG. 4) is in the direction of centrifugal force F, while the thicknessdirection of the bag (vertical direction in FIG. 4) is in the directionof rotation.

The rotary container 52 is provided with, for example, a pressingmechanism 53 for pressing the blood bag 10 from sides. The pressingmechanism 53 has, for example, as shown in FIG. 4, a pair of pressingplates 54 and 55 that sandwich the blood bag 10 from both sides. Onepressing plate 54 can be moved horizontally by a pressing actuator 56such as a cylinder toward the other pressing plate 55 to press the bloodbag 10 therebetween. Blood in the blood bag 10 can thus be pressed outtoward the first channel 13 or the second channel 14.

Next, a blood cell removal method using the cell removal system 1 thusconfigured will be described.

First, blood collected from the human body is introduced through thechannel 20 for blood collection and received in the blood bag 10. Atthis time, the first on-off valve 30 and the second on-off valve 31 areclosed. Next, the cell removal system 1 is mounted on the rotarycontainer 52 of the centrifugal separator 2. At this time, the blood bag10 is, for example, as shown in FIG. 4, installed between the pressingplate 54 and the pressing plate 55 in such a manner that the first endportion 10 a faces the side of the direction of centrifugal force F,while the second end portion 10 b faces the opposite side from thedirection of centrifugal force F.

Next, the centrifugal separator 2 operates such that the rotarycontainer 52 is rotated by the rotary actuator 51, whereby the blood inthe blood bag 10 is separated by centrifugal force, along the directionof centrifugal force F, into separation layers A, B, and C having aconcentration gradient of leukocyte cells, for example, and a separationlayer D as an additional separation layer as shown in FIG. 1. Theleukocyte cell concentration increases in order of separation layers C,B, and A (A<B<C), for example. Incidentally, the leukocyte cellconcentration of the separation layer D is lower than the separationlayer C. The separation layers A, B, and C are component layerscontaining a large amount of erythrocyte cells and serve as packed celllayers having a relatively high cell concentration. The separation layerD is a component layer containing plasma as a main component and servesas a sparse cell layer having a relatively low cell concentration. Inaddition, the red blood cell concentration increases in order ofseparation layers A, B, and C (A>B>C), and the separation layer Ccontains a large amount of platelets.

Next, the second on-off valve 31 is opened, and the pressing mechanism53 operates such that the blood bag 10 is pressed by the pressing plates54 and 55. Accordingly, the separation layer D that is close to thesecond end portion 10 b on the opposite side from the direction ofcentrifugal force F is pressed out from the blood bag 10, and sent tothe second blood component bag 12 through the second channel 14 shown inFIG. 1. Accordingly, a plasma component as a second body fluid componentis received in the second blood component bag 12. This serves as aplasma product.

After the entire separation layer D is pressed out from the blood bag10, next, the second on-off valve 31 is closed, and the first on-offvalve 30 is opened. Subsequently, the blood bag 10 is further pressed bythe pressing plates 54 and 55, and the separation layers A, B, and C arepressed out into the first channel 13 in order of closeness to the firstend portion 10 a. Accordingly, they are pressed out into the firstchannel 13 in ascending order of leukocyte cell concentration, that is,in the following order: separation layers A, B, and C. First, theseparation layer A having the lowest leukocyte cell concentration ispassed through the filter 15 for the removal of white blood cells, andreceived in the first blood component bag 11. Next, the separation layerB having the second lowest leukocyte cell concentration is passedthrough the filter 15 for the removal of white blood cells, and receivedin the first blood component bag 11. Finally, the separation layer Chaving the highest leukocyte cell concentration is passed through thefilter 15 for the removal of white blood cells, and received in thefirst blood component bag 11. In this manner, a packed red blood cellcomponent containing red blood cells as a main component is received asa first body fluid component in the first blood component bag 11. Thisserves as a red blood cell product. Subsequently, the first on-off valve30 is closed. The process of cell removal from blood is thus completed.

According to above embodiment, by centrifugal force, separation layersA, B, and C having a concentration gradient of white blood cells, whichare predetermined cells, are formed, and the separation layers A, B, andC are passed through the filter 15 in ascending order of white bloodcell concentration. Thus, the side having the highest white blood cellconcentration can be the last to pass through the filter 15.Accordingly, filtration can be continued while suppressing a decrease inthe white blood cell removal performance caused by a temporary decreasein the number of adsorption sites accompanying the progress offiltration, which is seen in the case where a body fluid componenthaving a uniform cell concentration is passed through a filter. As aresult, the efficiency of white blood cell removal by the filter 15 canbe increased.

Incidentally, although the separation layer D is pressed out from theblood bag 10 prior to the separation layers A to C in the aboveembodiment, it is also possible to press out the separation layer Dafter the separation layers A to C.

In addition, in the above embodiment, the first channel 13 is connectedto the first end portion 10 a of the blood bag 10 on the side of thedirection of centrifugal force F. Therefore, it is also possible to usethe centrifugal force of the centrifugal separator 2 to send theseparation layers A to C from the blood bag 10 to the first bloodcomponent bag 11. This allows the separation layers A to C to be passedthrough the filter 15 while reliably maintaining their separation state.In addition, the separation layers A to C can be passed through thefilter 15 during or immediately after the centrifugal separation thatseparates blood into the separation layers A to D, whereby the time ofthe cell removal process can be shortened.

In addition, in the above embodiment, it is also possible to use thecentrifugal force of the centrifugal separator 2 to send the separationlayer D to the second blood component bag 12. In such a case, as shownin FIG. 5, the second channel 14 is connected to the first end portion10 a of the blood bag 10. Then, by the centrifugal force of thecentrifugal separator 2, the separation layers A to C are sent to thefirst blood component bag 11 through the first channel 13, and then theseparation layer D is sent to the second blood component bag 12 throughthe second channel 14. In such a case, the need for a special device forpressing out the separation layers A to D, such as the pressingmechanism 53, is eliminated, whereby the cost of cell removal processcan be reduced. In addition, the supply to the blood component bags 11and 12 can be performed during or following the centrifugal separationof blood, whereby the time of cell removal process can be furthershortened.

In the above embodiment, for example, as shown in FIG. 6, it is alsopossible to provide a filter 80 in the second channel 14 to remove whiteblood cells, for example, as necessary. In such a case, white bloodcells can be removed from the separation layer D by the filter 80 whenthe separation layer D passes through the second channel 14. Therefore,in the case where the separation layer D contains white blood cells, aplasma product from which white blood cells have been removed can beproduced. Incidentally, this example is also applicable to the casewhere, as shown in FIG. 5, the second channel 14 is connected to thefirst end portion 10 a of the blood bag 10.

In the above embodiment, a preservation liquid for preserving theeventually produced red blood cell product may be added to theseparation layers A to C before being passed through the filter 15. Forexample, as shown in FIG. 7, a preservation liquid supply circuit 100for supplying a preservation liquid is provided in the first channel 13in a position closer to the blood bag 10 than the filter 15. Thepreservation liquid supply circuit 100 includes, for example, apreservation liquid bag 101, a preservation liquid supply channel 102that connects the preservation liquid bag 101 and the first channel 13,and an on-off valve 103 that opens and closes the channel 102. Forexample, when the separation layers A to C are pressed out from theblood bag 10, the on-off valve 103 is opened, and a preservation liquidis supplied from the preservation liquid bag 101 through thepreservation liquid supply channel 102 to the first channel 13.Accordingly, the preservation liquid is added to the blood components ofthe separation layers A to C flowing through the first channel 13, andthen the separation layers A to C pass through the filter 15.

According to this example, the preservation liquid can be added to thered blood cell product at the same time as the white blood cell removalprocess. In addition, the separation layers A to C are diluted with thepreservation liquid and thus more easily pass through the filter 15.Therefore, the burden on the filter 15 can be reduced. Accordingly, thelife of the filter 15 can be prolonged. Incidentally, this example isalso applicable to the case where, as shown in FIG. 5, the secondchannel 14 is connected to the first end portion 10 a of the blood bag10.

In the above embodiment, when the separation layers A to C are passedthrough the filter 15, the passing rate may be changed according to theleukocyte cell concentration. For example, in the case where theseparation layers A to C are pressed out toward the filter 15 by thepressing mechanism 53, the rate at which the blood bag 10 is pressed bythe pressing plates 54 and 55 of the pressing mechanism 53 may bechanged to change the passing rate of each of the separation layers A toC through the filter 15. In the case where the separation layers A to Care pressed out toward the filter 15 using the centrifugal force of thecentrifugal separator 2, the centrifugal force may be changed to changethe passing rate of each of the separation layers A to C through thefilter 15. For example, it is possible that the passing rate is set lowwhen the separation layer C having a high white blood cell concentrationpasses through the filter 15, the passing rate is set high when theseparation layer A having a low white blood cell concentration passesthrough the filter 15, and the passing rate is set medium when theseparation layer B having a medium white blood cell concentration passesthrough the filter 15. This allows white blood cells to be appropriatelyremoved from each of the separation layers A to C without putting toomuch burden on the filter 15.

Preferred embodiments of the present invention have been described abovewith reference to the accompanying drawings, but the invention is notlimited to these examples. It is obvious that a person skilled in theart can conceive of various modifications or amendments within theconcept defined in the claims, and they are naturally construed as beingwithin the technical scope of the present invention.

For example, the above embodiment has shown an example where the bloodbag 10 is installed in such a manner that the length direction of thebag (vertical direction in FIG. 1) is in the direction of centrifugalforce F when the cell removal system 1 is mounted on the centrifugalseparator 2. However, as shown in FIG. 8, it is also possible that theblood bag 10 is installed in such a manner that the thickness directionof the bag (horizontal direction in FIG. 8) is in the direction ofcentrifugal force F. In such a case, the first channel 13 and the secondchannel 14 are connected to one end portion of the blood bag 10 in thethickness direction. In addition, it is also possible that the pressingmechanism 53 is configured to press the blood bag 10 from sides in thethickness direction thereof.

In the above embodiment, the blood component that undergoes white bloodcell removal and is received in the first blood component bag 11 is ared blood cell product. However, it may also be a whole blood product, aplatelet product, an intermediate product thereof, etc. For example, inthe case of a platelet product, for example, separation layers C and Dcontaining platelets pass through the filter 15 via the first channel 13in order of separation layers D and C, and a platelet product isreceived in the first blood component bag 11.

In addition, in the case of a whole blood product, although the cellremoval system 1 or the like mentioned above may be used, it is alsopossible to use a cell removal system 110 that has no second channel 14or second blood component bag 12 but has only the first channel 13 andthe first blood component bag 11 as shown in FIG. 9. In such a case, allthe separation layers A to D pass through the filter 15 via the firstchannel 13 in order of separation layer D, C, B, and A, for example, anda whole blood product is received in the first blood component bag 11.

In the above embodiment, four separation layers are mainly present, andthree of the separation layers mainly pass through the filter 15 andhave a concentration gradient of white blood cells. However, the numberof layers is not limited thereto. In addition, cells to be removed arenot limited to white blood cells either, and may also be other cellssuch as platelets and red blood cells. Further, the filter 15 may alsobe capable of removing other disease-causing substances such parasitesin addition to pathogenic substances such as white blood cells. Inaddition, the body fluid from which cells are removed is blood in theabove embodiment, but the present invention is also applicable to thecases of other body fluids such as bone marrow and umbilical cord blood.

EXAMPLES

Hereinafter, the results of experiments for the evaluation of theremoval performance of a filter will be shown.

The method for preparing a filter used in the experiments is as follows.

(1) Production of Filter Medium:

The following polymer was used as a material for a filter.

Random polymer of 2-hydroxyethyl methacrylate (HEMA) anddimethylaminoethyl methacrylate (DM) (the molar ratio between HEMA andDM=97:3, hereinafter abbreviated as “HM-3”).

At a monomer concentration in ethanol of 1 mol/L, HM-3 was synthesizedby random polymerization at 60° C. for 8 hours in the presence of 0.005mol/L of 2,2′-azobis(2,4-dimethylvaleronitrile) (manufactured by WakoPure Chemical Industries, Ltd. trade name: V-65) as an initiator.

First, HM-3 was dissolved in a water/ethanol mixed solvent (the weightratio of water/ethanol=5/95) to prepare a 0.1 wt % solution. Thefollowing nonwoven fabrics A and B were impregnated with this solutionand, after the removal of excess liquid, vacuum-dried at 40° C. for 16hours.

A: Polyester nonwoven fabric having an average fiber diameter of 1.8 μmproduced by melt-blowing

B: Polyester nonwoven fabric having an average fiber diameter of 1.2 μmproduced by melt-blowing

(2) Production of Filter

In a container having an effective filtration area of 67 mm×67 mm, theHM-3-coated polyester nonwoven fabric having an average fiber diameterof 1.8 μm produced by melt-blowing was placed so that the pack densitywas 0.18 g/cm³ and the thickness was 1.5 mm, and similarly the polyesternonwoven fabric having an average fiber diameter of 1.2 μm was placedthereunder so that the pack density was 0.2 g/cm³ and the thickness was3 mm.

Example 1

The blood centrifugal filtration method of Example 1 using the abovefilter is as follows.

(1) From a healthy individual, 250 mL of fresh whole blood having addedthereto CPD (Citrate Phosphate Dextrose) was collected in a blood bag(hereinafter referred to as “whole blood”).

(2) The whole blood was transferred to a 250-mL bottle forcentrifugation, placed in a centrifugal cup, and then subjected tocentrifugation at 1250 G for 5 minutes. The bottle for centrifugationwas then taken out from the centrifugal cup.

(3) In the bottle, about 140 mL of a packed red blood cell layer andabout 110 mL of a platelet-rich plasma layer had been formed.

(4) About 110 mL of the platelet-rich plasma layer was sucked andrecovered in a bottle. At this time, suction was performed carefully inorder not to affect the interface with the packed red blood cell layer.

(5) Then, 35 mL of the packed red blood cell layer remaining in thebottle was carefully collected by suction from the upper part to give afirst layer of the packed red blood cell fluid. The concentration ofwhite blood cells (measured using a microcell counter) was 469×10e²/μL,the hematocrit was 30.5%, and the concentration of platelets was29.1×10e⁴/μL.

(6) Further, 35 mL of the packed red blood cell layer remaining in thebottle was carefully collected by suction from the upper part to give asecond layer of the packed red blood cell fluid. The concentration ofwhite blood cells was 141×10e²/μL, the hematocrit was 53.5%, and theconcentration of platelets was 9.1×10e⁴/μL.

(7) Next, 30 mL of the packed red blood cell layer remaining in thebottle was collected from the upper part and mixed with 5 mL of a SAGMliquid (red blood cell preservation liquid) to give 35 mL of a thirdlayer of the packed red blood cell fluid. The concentration of whiteblood cells was 22×10e²/μL, the hematocrit was 60.2%, and theconcentration of platelets was 4×10e⁴/μL. Incidentally, from thedilution ratio, before the addition of the SAGM liquid, the white bloodcell concentration was 26×10e²/μL, the hematocrit was 70.2%, and theplatelet concentration was 5×10e⁴/μL.

(8) Next, 26 mL of the packed red blood cell layer remaining in thebottle was collected from the upper part and mixed with 9 mL of a SAGMliquid to give 35 mL of a fourth layer of the packed red blood cellfluid. The concentration of white blood cells was 9.3×10e²/μL, thehematocrit was 61.3%, and the concentration of platelets was 2×10e⁴/μL.Incidentally, from the dilution ratio, before the addition of the SAGMliquid, the white blood cell concentration was 12.5×10e²/μL, thehematocrit was 82.5%, and the platelet concentration was 3×10e⁴/μL.

(9) Next, 18 mL of the packed red blood cell layer remaining in thebottle was collected from the upper part and mixed with 8 mL of a SAGMliquid to give 26 mL of a fifth layer of the packed red blood cellfluid. The concentration of white blood cells was 7.6×10e²/μL, thehematocrit was 60.5%, and the concentration of platelets was 1×10e⁴/μL.Incidentally, from the dilution ratio, before the addition of the SAGMliquid, the white blood cell concentration was 11.0×10e²/μL, thehematocrit was 87.4%, and the platelet concentration was 1×10e⁴/μL.

(10) A five-port flexible pipe made of polyvinyl chloride was connectedto the inlet side of the white blood cell removal filter. A similar pipehaving a blood bag for recovery connected to its end was connected tothe outlet side of the filter. The first to fifth layers of the packedred blood cell fluid were transferred to five 50-mL-volume syringes,respectively, and the syringes were connected to five connecting portsof the pipe, respectively. The syringes were subjected to thoroughmixing by inversion so that the packed red blood cell fluid in eachsyringe was made uniform, and then installed in a syringe pump set at aflow rate of 20 mL/min.

(11) The syringe pump was switched on for the syringe containing thefifth layer of the packed red blood cell fluid, whereby the fifth layerwas sent to start filtration through the filter. When the fifth layer ofthe packed red blood cell fluid disappeared from the syringe, then thefourth layer of the packed red blood cell fluid was sent. Subsequently,the third layer, the second layer, and the first layer of the packed redblood cell fluid were sequentially sent. In this manner, in order fromthe fifth layer having a low white blood cell concentration to the firstlayer having a high white blood cell concentration, the layers weresent, passed through the filter, and recovered in the blood bag forrecovery.

(12) The amount of the packed red blood cell fluid eventually recoveredin the blood bag for recovery was 145 mL. Sampling after mixing showedthat the concentration of white blood cells (counted using a flowcytometer) was 3.01 cells/μL and the hematocrit was 57.6%, while noplatelets were detected. The total number of white blood cells in theeventually recovered 145 mL of the packed red blood cell fluid that hadundergone white blood cell removal was 0.44×10e⁶.

Comparative Example 1

The blood centrifugal filtration method of Comparative Example 1 usingthe above filter is as follows.

(1) From a healthy individual, 250 mL of whole blood having addedthereto CPD was collected in a blood bag.

(2) The whole blood was transferred to a 250-mL bottle forcentrifugation, placed in a centrifugal cup, and then subjected tocentrifugation at 1250 G for 5 minutes. The bottle for centrifugationwas then taken out from the centrifugal cup.

(3) In the bottle, about 140 mL of a packed red blood cell layer andabout 110 mL of a platelet-rich plasma layer had been formed.

(4) About 110 mL of the platelet-rich plasma layer was sucked andrecovered in a bottle. At this time, suction was performed carefully inorder not to affect the interface with the packed red blood cell layer.

(5) A 22-mL quantity of a SAGM liquid was added to the packed red bloodcell layer remaining in the bottle and then uniformly mixed to prepare162 mL of a packed red blood cell fluid. The concentration of whiteblood cells was 132×10e²/μL, the hematocrit was 53.7%, and theconcentration of platelets was 27.3×10e⁴/μL. As a result of calculation,the total number of white blood cells in 162 mL of the prepared packedred blood cell fluid was 2.1×10e⁹.

(6) The packed red blood cell fluid was dispensed into four 35-mLsyringes and one 22-mL syringe. A five-port flexible pipe made ofpolyvinyl chloride was connected to the inlet side of a white blood cellremoval filter produced in the same manner as in Example 1. A similarpipe having a blood bag for recovery connected to its end was connectedto the outlet side. Subsequently, the syringes were connected to fiveconnecting ports of the pipe, respectively. The syringes were subjectedto thorough mixing by inversion so that the packed red blood cell fluidin each syringe was made uniform, and then installed in a syringe pumpset at a flow rate of 20 mL/min. The white blood cell concentration ofthe packed red blood cell fluid is the same in all the syringes.

(7) The syringe pump was switched on for the first syringe containingthe packed red blood cell fluid, whereby the packed red blood cell fluidwas sent to start filtration through the filter. When the packed redblood cell fluid disappeared from the first syringe, then the sending ofthe fluid in the second syringe was started. In this manner, the packedred blood cell fluid of each of the five syringes was passed through thefilter and recovered in the blood bag for recovery.

(8) The amount of the packed red blood cell fluid eventually recoveredin the blood bag for recovery was 145 mL. Sampling after mixing showedthat the concentration of white blood cells (counted using a flowcytometer) was 7.46 cells/μL and the hematocrit was 53.7%, while noplatelets were detected. The total number of white blood cells in theeventually recovered 145 mL of the packed red blood cell fluid that hadundergone white blood cell removal was 1.08×10e⁶.

Comparative Example 2

The blood centrifugal filtration method of Comparative Example 2 usingthe above filter is as follows.

(1) From a healthy individual, 250 mL of whole blood having addedthereto to CPD was collected in a blood bag.

(2) The whole blood was transferred to a 250-mL bottle forcentrifugation, placed in a centrifugal cup, and then subjected tocentrifugation at 1250 G for 5 minutes. The bottle for centrifugationwas then taken out from the centrifugal cup.

(3) In the bottle, about 140 mL of a packed red blood cell layer andabout 110 mL of a platelet-rich plasma layer had been formed.

(4) About 110 mL of the platelet-rich plasma layer was sucked andrecovered in a bottle. At this time, suction was performed carefully inorder not to affect the interface with the packed red blood cell layer.

(5) Then, 35 mL of the packed red blood cell layer remaining in thebottle was carefully collected by suction from the upper part to give afirst layer of the packed red blood cell fluid. The concentration ofwhite blood cells (measured using a microcell counter) was 469×10e²/μL,the hematocrit was 30.5%, and the concentration of platelets was29.1×10e⁴/μL.

(6) Further, 35 mL of the packed red blood cell layer remaining in thebottle was carefully collected by suction from the upper part to give asecond layer of the packed red blood cell fluid. The concentration ofwhite blood cells was 141×10e²/μL, the hematocrit was 53.5%, and theconcentration of platelets was 9.1×10e⁴/μL.

(7) Next, 30 mL of the packed red blood cell layer remaining in thebottle was collected from the upper part and mixed with 5 mL of a SAGMliquid (red blood cell preservation liquid) to give 35 mL of a thirdlayer of the packed red blood cell fluid. The concentration of whiteblood cells was 22×10e²/μL, the hematocrit was 60.2%, and theconcentration of platelets was 4×10e⁴/μL. Incidentally, from thedilution ratio, before the addition of the SAGM liquid, the white bloodcell concentration was 26×10e²/μL, the hematocrit was 70.2%, and theplatelet concentration was 5×10e⁴/μL.

(8) Next, 26 mL of the packed red blood cell layer remaining in thebottle was collected from the upper part and mixed with 9 mL of a SAGMliquid to give 35 mL of a fourth layer of the packed red blood cellfluid. The concentration of white blood cells was 9.3×10e²/μL, thehematocrit was 61.3%, and the concentration of platelets was 2×10e⁴/μL.Incidentally, from the dilution ratio, before the addition of the SAGMliquid, the white blood cell concentration was 12.5×10e²/μL, thehematocrit was 82.5%, and the platelet concentration was 3×10e⁴/μL.

(9) Next, 18 mL of the packed red blood cell layer remaining in thebottle was collected from the upper part and mixed with 8 mL of a SAGMliquid to give 26 mL of a fifth layer of the packed red blood cellfluid. The concentration of white blood cells was 7.6×10e²/μL, thehematocrit was 60.5%, and the concentration of platelets was 1×10e⁴/μL.Incidentally, from the dilution ratio, before the addition of the SAGMliquid, the white blood cell concentration was 11.0×10e²/μL, thehematocrit was 87.4%, and the platelet concentration was 1×10e⁴/μL.

(10) A five-port flexible pipe made of polyvinyl chloride was connectedto the inlet side of the white blood cell removal filter. A similar pipehaving a blood bag for recovery connected to its end was connected tothe outlet side of the filter. The first to fifth layers of the packedred blood cell fluid were transferred to five 50-mL-volume syringes,respectively, and the syringes were connected to five connecting portsof the pipe, respectively. The syringes were subjected to thoroughmixing by inversion so that the packed red blood cell fluid in eachsyringe was made uniform, and then installed in a syringe pump set at aflow rate of 20 mL/min.

(11) The syringe pump was switched on for the syringe containing thefirst layer of the packed red blood cell fluid, whereby the first layerwas sent to start filtration through the filter. When the first layer ofthe packed red blood cell fluid disappeared from the syringe, then thesecond layer of the packed red blood cell fluid was sent. Subsequently,the third layer, the fourth layer, and the fifth layer of the packed redblood cell fluid were sequentially sent. In this manner, in order fromthe first layer having a high white blood cell concentration to thefifth layer having a low white blood cell concentration, the layers weresent, passed through the filter, and recovered in the blood bag forrecovery.

(12) The amount of the packed red blood cell fluid eventually recoveredin the blood bag for recovery was 145 mL. Sampling after mixing showedthat the concentration of white blood cells (counted using a flowcytometer) was 38.2 cells/μL and the hematocrit was 58.7%, while noplatelets were detected. The total number of white blood cells in theeventually recovered 145 mL of the packed red blood cell fluid that hadundergone white blood cell removal was 5.54×10e⁶.

The present invention is useful in improving the efficiency ofpredetermined cell removal by a filter in the separation of apredetermined body fluid component from a body fluid.

-   -   1: Cell removal system    -   2: Centrifugal separator    -   10: Blood bag    -   10 a: First end portion    -   10 b: Second end portion    -   11: First blood component bag    -   12: Second blood component bag    -   13: First channel    -   14: Second channel    -   15: Filter    -   30: First on-off valve    -   31: Second on-off valve    -   A to D: Separation layer    -   F: Direction of centrifugal force

We claim:
 1. A method for removing predetermined cells from a body fluid, the method comprising: applying centrifugal force to the body fluid placed in a first bag by centrifugal separation to form separation layers having a concentration gradient of the predetermined cells, providing a force generating member that comprises: at least one of a centrifugal separator that produces the centrifugal force and a press that applies a pressure to the first bag; passing the separation layers through a filter in ascending order of the concentration of the predetermined cells according to the concentration gradient of the predetermined cells to remove the predetermined cells from the separation layers; and receiving the separation layers from which the predetermined cells have been removed in a second bag; wherein, when the separation layers are passed through the filter, one of the centrifugal force and the pressure generated by the force generating member is changed so as to regulate and change a passing rate of each of the separation layers being passed through the filter according to a concentration of the predetermined cells in the separation layers having the concentration gradient of the predetermined cells.
 2. The method for removing predetermined cells according to claim 1, wherein a predetermined body fluid component produced by removing the predetermined cells from the separation layers is received in the second bag, and a preservation liquid for preserving the body fluid component is added to the separation layers before being passed through the filter.
 3. The method for removing predetermined cells according to claim 1, wherein an additional separation layer, other than the separation layers, is separated by the centrifugal force and is received in a third bag separately from the separation layers.
 4. The method for removing predetermined cells according to claim 1, wherein the predetermined cells are pathogens.
 5. The method for removing predetermined cells according to claim 1, wherein the body fluid is blood, the predetermined cells are white blood cells, and the predetermined cells are removed from the separation layers to produce a blood component containing red blood cells as a main component.
 6. The method for removing predetermined cells according to claim 1, wherein the one of the centrifugal force and pressure is regulated such that the passing rate of the separation layer having a relatively high concentration of the predetermined cells is set relatively low and the passing rate of the separation layer having a relatively low concentration of the predetermined cells is set relatively high. 